Intersectional strategy to study cortical inhibitory parvalbumin-expressing interneurons

Parvalbumin-expressing (PV) interneurons are key neuronal elements to a global excitatory-inhibitory balance in normal cortical functioning. To better understand the circuit functions of PV interneurons, reliable animal models are needed. This study investigated the sensitivity and specificity of the most frequently used PV-Cre/tdTomato mouse line in this regard. The colocalization of the transgene (tdTomato) with the parvalbumin protein, with GAD1 (a conclusive inhibitory cell marker) and Vglut1 (a conclusive excitatory cell marker) as well as with a marker for perineuronal nets (WFA) was assessed and a substantial proportion of layer 5 PV neurons was found to be excitatory and not inhibitory in the PV-Cre/tdTomato mouse. The intersectional transgenic mouse line Vgat-Cre/PV-Flp/tdTomato provided a solution, since no colocalization of tdTomato with the Vglut1 probe was found there. In conclusion, the Vgat-Cre/PV-Flp/tdTomato mouse line seems to be a more reliable animal model for functional studies of GABAergic PV interneurons.

We defined colocalization as the overlap of the red fluorescent label, AlexaFluor 594 (corresponding to the genetically encoded tdTomato signal) with the green fluorescent label, AlexaFluor 488 (corresponding to parvalbumin antibody labeling) and used it as an assay to probe the specificity of Cre expression for parvalbuminexpressing neurons in this mouse line (n = 7 brain sections from 4 mice) (Fig. 1a).At first, we studied how many of the red fluorescent cells also have a green labeling to probe how specific tdTomato fluorescence is for parvalbumin-expressing cells in this mouse line.In 1 mm 3 barrel cortex, there were 7846.6 ± 816.0 cells/mm 3 showed a colocalization (white arrows in Fig. 1d) of tdTomato and parvalbumin, which resulted in a mean colocalization of 87.6 ± 6.7%.Furthermore, 348.4 ± 279.2 cells showed only parvalbumin expression (green arrowheads in Fig. 1b,c) and 1097.0 ± 561.8 cells showed only tdTomato expression (red arrowheads in Fig. 1b,c).Layer I was not considered in the analysis due to low cell body numbers (being in agreement with a virtual absence of PV cells in layer 1) 38 .The layer-specific distribution of cells showing a colocalization of the red and green fluorescent labels had the highest proportion in layer 2/3 (mean: 90.8 ± 8.0%) and the lowest in layer 5b (mean: 81.2 ± 8.3%) (Fig. 1e).The highest number of colocalized cells was observed in layer 4 (mean: 2357.7 ± 394.2, median: 2390.1, 25% percentile: 2111.2, 75% percentile: 2560.1, min: 1711.5, max: 2983.3), the lowest in layer 6 (mean: 1014.6 ± 299.0, median: 910.1, 25% percentile: 779.9, 75% percentile: 1269.3,min: 775.2, max: 1572.6).The highest number of non-colocalized cells was observed in layer 5b (mean: 423.1 ± 187.3, median: 430.3, 25% percentile: 338.1, 75% percentile: 581.37, min: 107.7, max: 693.3).After examining the colocalization of tdTomato with parvalbumin, we took the reverse perspective and observed the colocalization of the green fluorescent label with the red signal in our target cells (orange columns in Fig. 1f).The mean colocalization of parvalbumin antibody labeling with tdTomato expression as a measure of sensitivity corresponded to 95.6 ± 3.7% in the overall cortex.
These results suggest that the mouse model is sensitive, but its specificity needs to be discussed further (see Discussion "Technical considerations").Since previously some weakly PV-expressing excitatory neurons had been described 19 , we next examined the potential excitatory nature of tdTomato-expressing cells, while at the same time verifying their assumed inhibitory phenotype.Toward this aim, the colocalization of tdTomato with inhibitory and excitatory cell markers was observed.We used the Gad1 probe (glutamic acid decarboxylase 67 kD, the key enzyme necessary for GABA synthesis) as a marker for inhibitory cells and the Vglut1 probe (vesicular glutamate transporter 1, the synaptic vesicle transporter of glutamate) as a marker for excitatory cells.
These results predict that in the PV-Cre mouse line, any transgene expression will not be restricted to GABAergic interneurons but will also involve excitatory neurons mainly in layer 5b but also in layer 5a.

Characterization of cortical parvalbumin-expressing neurons in the Vgat-Cre/PV-Flp mouse line
As a possible solution for obtaining a tool to exclusively study GABAergic PV cells, we investigated the colocalization profile of tdTomato-expressing cells in the Vgat-Cre/PV-Flp mouse using altogether 4 mice.In this mouse line, the transgene should be expressed only in GABAergic cells.In the Vgat-Cre mouse, we tested the layer-specific distribution of tdTomato-expressing cells on 59 brain sections.The highest number of tdTomato-expressing cells was found in layer 4 (2532.9± 645.9 cells/mm 3 ), which corresponds to a proportion of 29.7 ± 7.0% of all tdTomato-expressing cells.The lowest number of tdTomato-expressing cells was observed in layer 5a (1267.8± 351.1 cells/mm 3 ), corresponding to a proportion of 14.8 ± 3.7%.Layer 5b contained 1822.2 ± 517.7 cells/mm 3 , corresponding to 21.2 ± 5.3%.Layers 2/3 and 6 contained 1365.1 ± 505.7 cells/mm 3 and 1582.9 ± 465.3 cells/mm 3 resp.(Supplementary Fig. 2).
These results imply that the Vgat-Cre/PV-Flp mouse line is highly specific for targeting transgene expression to GABAergic parvalbumin-expressing interneurons.

Perineuronal nets
For this part of the study, we wanted to leverage the known tight association of perineuronal nets with PV neurons [39][40][41][42][43] to inquire whether they have a potential to distinguish between inhibitory and excitatory PV neurons.Thus, we compared the PNN-enwrapping of tdTomato-cells in both mouse lines.For this purpose, we used WFA (Wisteria floribunda agglutinin), a lectin labeling chondroitin sulfate proteoglycans as a major component of the PNN 25,44,45 .

Discussion
In contrast to many previous BAC transgenic mice 46,47 , IRES-Cre knock-in mice like PV-Cre are generally considered to have a highly specific transgene expression with respect to the targeted cell population 5,48 .However, once in a while, at least for some brain areas, off-target expression 10 or low sensitivity 11 has been reported.This points to the prevailing need to characterize each transgenic mouse line for specificity and sensitivity of its transgene expression, in order to be able to draw firm conclusions from functional manipulation experiments.
Parvalbumin (PV)-expressing GABAergic interneurons have a crucial role in maintaining the excitationinhibition balance in the cortex and contribute to the development of psychiatric conditions like autism, schizophrenia or Alzheimer`s disease [49][50][51][52] .Therefore, it is very important to find reliable animal models specific and sensitive for this cell type.In the present study, we compared the widely used PV-Cre/tdTomato and the more recently introduced Vgat-Cre/PV-Flp/tdTomato mouse lines with regard to specificity of genetically targeted recombinase expression for PV-expressing interneurons.We show that only with an intersectional approach 14 the physiologically occurring population of PV-expressing pyramidal cells in L5 3,19 can be excluded from functional manipulations like optogenetics or chemogenetics, in order to achieve an unbiased readout.

Technical considerations
Ideally, one would like to have a highly sensitive and specific transgenic animal model, which labels 100% of all target cells as verified by protein or mRNA detection of a marker molecule that characterizes the neurons of interest.However, due to inherent technical limitations caused by histological processing, neither immunohistochemistry nor in situ hybridization (ISH) does achieve this performance.Antibodies can suffer from fixation masking epitopes or from poor penetration 53,54 .In the same direction, RNA probes are sensitive to a number of experimental factors that might compromise the readout 55 .As we also used lectin histochemistry, here the sugar moieties that are detected by a single lectin may not be inclusive for all molecular compositions of perineuronal nets that do exist 23 .
What other reasons could explain the roughly 12-13% of "unidentified" tdTomato cells in both mouse lines?One possibility could be that they underwent Cre-mediated recombination during early development but later in life lost their ability to express parvalbumin due to unknown molecular biological mechanisms.A similar phenomenon was suggested by Hu et al. 9 for the SOM-Cre mouse line.
Another explanation could be the activity-dependent expression of the parvalbumin protein.Donato et al. have observed two populations of parvalbumin-expressing cortical interneurons: cells with a low parvalbuminexpression and a low excitatory-inhibitory synaptic density ratio and cells with a high parvalbumin-expression and a high excitatory-inhibitory synaptic density ratio 56 .According to this phenomenon, cells expressing the tdTomato signal which are negative for the parvalbumin antibody may be parvalbumin-expressing cells having a low protein expression due to low activation and therefore escape detection by the antibody labeling.This could also explain, why we still have ~ 12-13% of tdTomato + /aPV-cells in the Vgat-Cre/PV-Flp mouse line where the parvalbumin-expressing excitatory cells are eliminated.Furthermore, Filice et al. have shown, that in ASD mouse models, the number of parvalbumin interneurons is unchanged, while parvalbumin protein and PValb mRNA levels decrease, proving the existence of parvalbumin-expressing cells with a very low protein expression level 57 .
We also want to state here that in years of studying tdTomato-labeled cells in the PV-Cre/tdTomato mouse line with patch clamp recordings and subsequent single cell reconstruction, all neurons in all layers were fast spiking and showed a basket-cell like morphology 58

(Preuss et al., unpublished observations).
Keeping this in mind, we will now carefully consider the results of our colocalization studies.

Colocalization of the tdTomato signal with the parvalbumin antibody, glutamic acid decarboxylase 67 (Gad1) probe and vesicular glutamate transporter 1 (Vglut1) probe
In the PV-Cre/tdTomato mouse, the mean colocalization ratio of tdTomato with parvalbumin corresponded to 87.6 ± 6.7%.The mean colocalization of parvalbumin with tdTomato to 95.6 ± 3.7%.Assuming that ~ 12% of tdTomato-labeled neurons that did not express parvalbumin are a consequence of too low sensitivity of the antibody, we tentatively conclude that this mouse line is sensitive and specific for cortical PV neurons.To further investigate the inhibitory or potential excitatory nature of the tdTomato-expressing cells, their colocalization with Gad1 and Vglut1, respectively, was studied.The mean colocalization of tdTomato with Gad1 corresponded to 92.1 ± 3.3%, the mean colocalization of tdTomato with Vglut1 to 5.3 ± 1.6%, pointing to a small but reliable excitatory neuron population.Interestingly, the highest number of tdTomato/Vglut1 colocalizing cells and the highest colocalization ratio was found in layer 5b (414.7 ± 1885.3 cells/mm 3 and 18.2 ± 6.6% resp.), which is consistent with previous findings describing parvalbumin-expressing cells, that are negative for GABA 59 and found to be pyramidal cells 19 .Our study now provides a quantification of the proportion of these parvalbumin-expressing excitatory cells (5.3 ± 1.6%) averaged across all layers and revealed their-specific distribution by showing, that most of these cells are in layers 5a (6.0 ± 4.7%) and 5b (18.2 ± 6.6%).
In Vgat-Cre/PV-Flp/tdTomato mice, the mean colocalization of tdTomato with parvalbumin corresponded to 87.8 ± 6.6% and the mean colocalization of parvalbumin with tdTomato to 97.6 ± 1.0%.These figures, under the same assumption as above, also lead to the conclusion that this mouse line is sensitive and specific for PV cells.But did it also rid us from the excitatory cell component?
The colocalization of tdTomato and Vglut1 corresponded to 0.2 ± 0.4% (versus 98.9 ± 1.4% colocalization with Gad1), so we have good evidence that, in this intersectional mouse line, the pyramidal neurons coexpressing parvalbumin were excluded from tdTomato expression.This is due to the molecular biological features of the Vgat-Cre/PV-Flp/tdTomato mouse line, which equal a biological "and"-gate 60 .
Another matter of caveat is the fact, that we used predominantly male animals in this study.Retrospectively, we consider this as scientifically undesirable.However, studies have shown that for histology data there were no www.nature.com/scientificreports/sex differences and that females were not more variable than males 61 .Other studies have shown that the variability of gene expression was similar in males and females 62 .The predominance of male animals in our study had therefore most probably not much, if any, impact on our results.

Codistribution with WFA
Perineuronal nets (PNNs) are part of the extracellular matrix surrounding the cell bodies and proximal dendrites of certain neurons 23 .Thus, the term colocalization here means codistribution in the immediate vicinity if the target cell`s plasma membrane.In different regions of the central nervous system, the cell types surrounded by PNNs are different: in the neocortex, mainly the parvalbumin-expressing GABAergic interneurons are surrounded by the PNNs 43,63 .However, in certain regions of the hippocampus 64 , PNNs surround excitatory and inhibitory cells.
In the neocortex of the mouse, the parvalbumin-expressing neurons are the main cell type surrounded by perineuronal nets 23 .This is consistent with our findings, since the mean colocalization of WFA with tdTomato corresponded to 99.2 ± 0.9% in the PV-Cre/tdTomato mouse and to 97.8 ± 1.2% in the Vgat-Cre/PV-Flp/ tdTomato mouse.The mean colocalization of tdTomato with WFA corresponded to 70.9 ± 5.6% in the PV-Cre/ tdTomato mouse and to 80.7 ± 5.5% in the Vgat-Cre/PV-Flp/tdTomato mouse, which shows a significant difference (p = 0.003).Thus, the large majority of the PV-cells have a PNN-ensheathment.Those that do not could be chandelier cells, which also would explain the pronounced non-colocalization in layer 2/3, which is home to many of these cells 65 .Altogether, our visualization of PNNs did not disclose a means to distinguish between GABAergic and glutamatergic PV neurons.
However, since not every type of perineuronal net can be visualized with WFA 66 , future studies using different lectins or antibodies might reveal strategies that could substitute for the laborious intersectional breeding strategy.
Summing up the results, our study has shown that the Vgat-Cre/PV-Flp/tdTomato mouse line is a more reliable animal model for experiments investigating the synaptic connections and functions of parvalbumin-expressing GABAergic interneurons 6,8 , since it rules out layer 5 pyramidal cells coexpressing parvalbumin.Future studies using the PV-Cre/tdTomato mouse line have to consider L5 pyramidal cells as a putative confounder.

Methods
All experiments were carried out according to the German guidelines of animal care.The experimental protocol was approved by the Niedersächsische Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES; 33.19-42502-04-15/1897).The animals were kept under a 12 h day/12 h night cycle and had access to food and water ad libitum.
All mice were transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4) after an intraperitoneal injection of ketamine (0.1 ml/10 g bodyweight).After the perfusion, the brains were removed from the skull and underwent postfixation (2 h in PFA at 4 °C).The brains used for immunohistochemistry were washed 2 × 15 min in 0.1 M PB pH 7.4 and stored at 4 °C until cutting.The brains for fluorescent in situ hybridization (FISH) were immersed in a cryoprotective solution (20% sucrose solution in 0.01 M PB with 0.9% NaCl, PBS, pH 7.4), stored overnight at 4 °C and were shockfrozen with isopentane at − 40 °C and stored at − 80 °C until cutting.

Parvalbumin-immunohistochemistry and lectin histochemistry
The brains were cut in 40 µm-thick sections through the barrel cortex with a vibratome (VT 1200S, Leica, Wetzlar, Germany).The sections were washed 2 × 15 min in 0.05 M Tris buffer (TB, pH 7.6), 2 × 15 min 0.05 M Tris-buffered saline (TBS) pH 7.6 and 2 × 15 min with TBS and 0.5% Triton X-100 (TBST, pH 7.6) at room temperature.The sections were then blocked with 0.25% bovine serum albumin (BSA; Carl Roth, Karlsruhe, Germany) and 10% normal goat serum (NGS; Jackson Immuno-Research Laboratories, West Grove, Pennsylvania, USA) in TBST (0.05 M, pH 7.6).The primary antibody rabbit-anti-PV 25 (Swant, Martly, Switzerland) and the biotinylated lectin Wisteria floribunda agglutinin (WFA; Sigma-Aldrich, Munich, Germany) were diluted in the blocking solution to 1:5000 and 1:2000, respectively.Three brain sections were stained for each label, with the sections being incubated for 72 h at 4 °C.The sections were then washed 4 × 15 min with TBST (0.05 M, pH 7.6) at room temperature.After washing, the sections were incubated in the secondary antibody, goat-anti-rabbit Alexa 488 (Life Technologies, Darmstadt, Germany) diluted 1:500 in TBST (0.05 M, pH 7.6) or in Streptavidin Alexa Fluor 488 (Thermo Fisher Scientific, Massachusetts, USA) diluted 1:300 in TBST (0.05 M, pH 7.6) for 4 h at room temperature.The sections were washed after this incubation for 2 × 15 min in TBST (0.05 M, pH 7.6) and 1 × 15 min in TBS (0.05 M, pH 7.6) at room temperature.To display the cell nuclei, sections were also stained with 4′-5-diamidino-2-phenylindole (DAPI, Life Technologies, Darmstadt, Germany) diluted 1:5000 in TBS (0.05 M, pH 7.6) for 5 min at room temperature.After staining the nuclei, the sections were washed 1 × 15 min Vol:.(1234567890 On the following days, the sections were washed in diluted SSC and a solution of diluted SSC and 50% formamide.Then, a solution of RNAse (4 µg/mL, Marcherey-Nagel, Düren, Germany) and 2 × diluted SSC was applied to get rid of residual RNA.Afterwards, the sections were once again washed with 2 × diluted SSC and a solution of SSC and 50% formamide.Afterwards, the sections were blocked in 4% normal goat serum in TBS (0.05 M, pH 7.5) at room temperature.After blocking, the sections were washed in TBS (0.05 M, pH 7.5) for 3 × 2 min at room temperature and 0.5% blocking solution (TSA Biotin System, Akoya Biosciences, Marlborough, Massachusetts, USA) was applied for 1 × 60 min at room temperature.The sections were then incubated in peroxidase-conjugated anti-digoxigenin (1:2000 in 0.5% blocking solution) at 4 °C overnight.
Since the FISH protocol completely quenched the tdTomato fluorescence, an immunohistochemical staining was performed to recover the native signal.The sections were washed 3 × 15 min in TBS pH 7.5 and blocked for 90 min with 10% normal goat serum in TBS pH 7.5.The primary antibody rabbit-anti-RFP (Rockland, Philadelphia, Pennsylvania, USA) was diluted in the blocking solution to 1:500, incubated for 72 h at 4 °C.The sections were then washed 4 × 15 min with TBS pH 7.5 at room temperature.After washing, the sections were incubated in the secondary antibody, goat-anti-rabbit Alexa 594 (Life Technologies, Darmstadt, Germany) diluted 1:500 in TBS pH 7.5 for 4 h at room temperature.The sections were washed after this incubation for 3 × 10 and 3 × 20 min in TBS pH 7.5 at room temperature.To display the cell nuclei, sections were stained with 4'-5-diamidino-2-phenylindole (DAPI) diluted 1:1000 in TBS pH 7.5 for 5 min at room temperature.After staining the nuclei, the sections were washed 3 × 15 and 2 × 15 min in TBS pH 7.5.Then the sections were mounted on slides and covered with coverslips coated with Aqua Poly-Mount.

Image acquisition and data analysis
Analysis and cell counting on brain sections was carried out with a fluorescence microscope (AxioImager.M2, Zeiss, Jena, Germany) with structured illumination (ApoTome, Zeiss).Cell counting was performed blinded.Further images were taken with the confocal microscope (Zeiss LSM 880) for illustration.For each probe or antibody, a 3D-images stack of the barrel cortex, identified by DAPI staining with a 25 × objective at an optical z-resolution of 1 µm were taken.In this 3D image, a 1000 µm-wide area through the barrel cortex was chosen for analysis and the cells were counted with Neurolucida (MBF Bioscience, Williston, Vermont, USA).Fluorescentlylabeled cells of interest in this area were counted in a user-dependent manner if they were completely or (at the border) more than 50% within the delineated area.The DAPI staining enabled to delineate the cortical layers according to standard cytoarchitectonic criteria.The layer-specific analysis of the counted cells was carried out by Neuroexplorer (MBF Bioscience).The program reports the number of counted cells according to their defined markers and calculated the area of each layer and the total area in the chosen area of the barrel cortex in µm 2 .The calculated cell numbers were normalized in each section to 1 mm 3 barrel cortex as described in 12 .In brief, first we multiplied the size of the total area (µm 2 ) by the thickness of the Sect.(40 µm).This volume (in µm 3 ) was scaled-up to 1 mm 3 barrel cortex using a normalization factor, which was applied to the counted cell numbers in the total area, which made cell quantification from individual section comparable to each other.The same factor was also used to extrapolate the number of cells for each layer individually, so that the resulting values reflect the number of cells in their respective layer in a volume of 1 mm 3 cortex (number of cell/mm 3 ), to allow for a comparison across samples.A total of 26,014 cells were counted.

Figure 1 .
Figure 1.Colocalization of parvalbumin and tdTomato in the barrel cortex of the PV-Cre/tdTomato mouse.(a) Overview image showing immunoamplified transgenic tdTomato and immunohistochemical staining for parvalbumin.Red corresponds to tdTomato-signal, green corresponds to parvalbumin antibody labeling.On the left margin, cell nuclei are shown in blue (DAPI) for laminar delineation; wm: white matter.The asterisk indicates axons of tdTomato cells (putative parvalbumin-expressing pyramidal cells) leaving the cortex.Scalebar: 200 µm.(b-d) higher magnification of the boxed area in (a).(b) reveals the expression of parvalbumin (aPV, green), (c) shows the tdTomato signal (red) and (d) displays the colocalization of tdTomato and parvalbumin.The white arrows indicate colocalization, whereas green arrowheads label the cells expressing only parvalbumin protein and red arrowheads label the cells expressing only the tdTomato transgene.Scalebar: 30 µm.(e) Counts of colocalized (yellow; colocalization rate on top) and non-colocalized (green and red) cells in a layer-specific manner.(f) Colocalization rate of tdTomato with parvalbumin (indicating specificity, yellow) versus the colocalization rate of the parvalbumin antibody with tdTomato (indicating sensitivity, orange).

Figure 2 .
Figure 2. Colocalization of tdTomato with the inhibitory cell marker Gad1 and the excitatory cell marker Vglut1 within the barrel cortex of the PV-Cre/tdTomato mouse line.(a) Overview image showing transgene expression and in-situ hybridization for Gad1.Red corresponds to tdTomato signal, green corresponds to the Gad1 probe.On the left margin, cell nuclei are shown in blue (DAPI) for laminar delineation; wm: white matter.Scalebar: 200 µm.(b-d) Higher magnification of the boxed area in a) revealing a high but not thorough colocalization (d) of Gad1 (b) and tdTomato signal (c).Scalebar: 30 µm.(e) Overview image showing transgene expression and in-situ hybridization for Vglut1.Red corresponds to tdTomato-signal, green corresponds to the Vglut1 probe.On the left margin, cell nuclei are shown in blue (DAPI) for laminar delineation; wm: white matter.Scalebar: 200 µm.(f-h) Higher magnification of the boxed area in (e) revealing a low but not absent colocalization (h) of Vglut1 (f) and tdTomato signal (g).Scalebar: 30 µm.White arrows indicate colocalization, whereas green arrowheads label the cells expressing only the Gad1 (b and c) or the Vglut1 (f,g) probe.Please note, to keep Figs.4f and 4g clear, not every cell showing the Vglut1 probe was marked with a green arrowhead.Red arrowheads label the cells expressing only the tdTomato signal.(i) Cell counts of tdTomato/Gad1colocalizing neurons show a peak expression of non-colocalized and thus presumably excitatory cells in layer 5b.(j) Complementary cell counts of tdTomato/Vglut1-colocalizing neurons show a peak expression of colocalized and thus excitatory cells in layer 5b.The colocalization rate is indicated on each yellow column.

Figure 3 .
Figure 3. Colocalization of parvalbumin and tdTomato in the barrel cortex of the Vgat-Cre/PV-Flp/tdTomato mouse.(a) Overview image showing immunoamplified transgenic tdTomato and immunohistochemical staining for parvalbumin.Red corresponds to tdTomato signal, green corresponds to parvalbumin antibody labeling.On the left margin, cell nuclei are shown in blue (DAPI) for laminar delineation; wm: white matter.Scalebar: 200 µm.(b-d) higher magnification of the boxed area in (a).(b) reveals the expression of parvalbumin (green; aPV), (c) shows the tdTomato signal (red) and (d) displays the colocalization of tdTomato and parvalbumin.Scalebar: 30 µm.The white arrows indicate colocalization whereas green arrowheads label the cells expressing only parvalbumin protein and red arrowheads label the cells expressing only the tdTomato transgene.(e) Counts of colocalized (yellow; colocalization rate on top) and non-colocalized (green and red) cells in a layer-specific manner, applying PV immunohistochemistry. (f) Colocalization rate of tdTomato with the parvalbumin antibody (indicating specificity, yellow) versus the colocalization rate of the parvalbumin antibody with tdTomato (indicating sensitivity, orange).

Figure 4 .
Figure 4. Colocalization of tdTomato with the inhibitory cell marker Gad1 and the excitatory cell marker Vglut1 within the barrel cortex of the Vgat-Cre/PV-Flp/tdTomato mouse line.a) Overview image showing transgene expression and in-situ hybridization for Gad1.Red corresponds to tdTomato signal, green corresponds to the Gad1 probe.On the left margin, cell nuclei are shown in blue (DAPI) for laminar delineation; wm: white matter.Scalebar: 200 µm.b-d) Higher magnification of the boxed area in a) revealing a high but not thorough colocalization (d) of Gad1 (b) and tdTomato signal (c).Scalebar: 30 µm.e) Overview image showing transgene expression and in-situ hybridization for Vglut1.Red corresponds to tdTomato signal, green corresponds to the Vglut1 probe.On the left margin, cell nuclei are shown in blue (DAPI) for laminar delineation; wm: white matter.Scalebar: 200 µm.f-h) Higher magnification of the boxed area in e) revealing a virtually non-existent colocalization rate (h) of Vglut1 (f) and tdTomato signal (g).Scalebar: 30 µm.White arrows indicate colocalization, whereas green arrowheads label the cells expressing only the Gad1 (b and c) or the Vglut1 (f and g) probe.Red arrowheads label the cells expressing only the tdTomato signal.i) Cell counts of tdTomato/Gad1-colocalizing neurons show a nearly complete overlap of the two signals.j) Complementary cell counts of tdTomato/Vglut1-colocalizing neurons hardly show any overlap.The colocalization rate is indicated on each yellow column.

Figure 5 .
Figure 5. Colocalization of perineuronal nets and tdTomato in the barrel cortex of the PV-Cre/tdTomato mouse.(a) Overview image showing immunoamplified transgenic tdTomato and histochemical staining for WFA.Red corresponds to tdTomato signal, green corresponds to WFA labeling.On the left margin, cell nuclei are shown in blue (DAPI) for laminar delineation; wm: white matter.Scalebar: 200 µm.b-d) higher magnification of the boxed area in a).b) reveals the expression of WFA (green), c) shows the tdTomato signal and d) displays the colocalization of tdTomato and WFA.Scalebar: 30 µm.The white arrows indicate colocalization, whereas green arrowheads label the WFA-surrounding and red arrowheads label the cells expressing only the tdTomato transgene.e) Counts of colocalized (yellow; colocalization rate on top) and non-colocalized (green and red) cells in a layer-specific manner.f) Colocalization rate of tdTomato with WFA (yellow; indicating that not every PV cell possesses a perineuronal net) versus the colocalization rate of WFA with tdTomato (orange; indicating that all perineuronal nets were around PV cells).

Figure 6 .
Figure 6.Colocalization of perineuronal nets and tdTomato in the barrel cortex of the Vgat-Cre/PV-Flp/ tdTomato mouse.a) Overview image of immunoamplified transgenic tdTomato and histochemical staining for WFA.Red corresponds to tdTomato signal, green corresponds to WFA labeling.On the left margin, cell nuclei are shown in blue (DAPI) for laminar delineation; wm: white matter.Scalebar: 200 µm.(b-d) higher magnification of the boxed area in (a).b) reveals the expression of WFA (green), c) shows the tdTomato signal and d) displays the colocalization of tdTomato and WFA.Scalebar: 30 µm.The white arrows indicate colocalization, whereas green arrowheads label the WFA-surrounding and red arrowheads label the cells expressing only the tdTomato transgene.e) Counts of colocalized (yellow; colocalization rate on top) and non-colocalized (green and red) cells in a layer-specific manner.(f) Colocalization rate of tdTomato with WFA (yellow; indicating that not every PV cell possesses a perineuronal net) versus the colocalization rate of WFA with tdTomato (orange; indicating that all perineuronal nets were around PV cells). https://doi.org/10.1038/s41598-024-52901-y TBS (0.05 M, pH 7.6) and 2 × 15 min in TB (0.05 M, pH 7.6) and stored in TB (0.05 M, pH 7.6) overnight at 4 °C.Then the sections were mounted on slides and covered with coverslips coated with Aqua Poly-Mount (Polyscience, Warrington, Pennsylvania, USA).Mouse brains were stored at − 80 °C and cut into 40 µm-thick sections through the barrel cortex (Bregma − 1.06 mm to − 1.94 mm) with a cryostat (Leica CM3050 S, Nuβloch, Germany) at − 18 °C.Four PV-Cre/tdTomato and 4 Vgat-Cre/PV-Flp/tdTomato mouse brain sections were cut, collected in PBS (pH 7.4) and put in a multiwell-plate after washing with PBS (0.1 M, pH 7.4).To increase the permeability of the tissue, the sections were treated with 1% H 2 O 2 in methanol for 20 min, followed by quenching in 0.2 M HCl for 8 min and finally treated with proteinase K dissolved in Tris-HCl/EDTA (10 mg/mL) for 10 min.GCC TTG TCC CCG GTG TCA TA)) were prepared in the hybridization buffer and heat-shocked at 95 °C for 2 min.Later, the probes were added to the sections and the hybridization took place at 55 °C overnight.